1 Bio-inspired Antioxidant Defense System Constructed by

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Bio-inspired Antioxidant Defense System Constructed by Antioxidants-Eluting Electrospun F127-based Fibers Haozheng Wang, Xiaodong Xu, Runhai Chen, Jiruo Zhao, Lele Cui, Guangkuo Sheng, Qiang Shi, Shing-Chung Wong, and Jinghua Yin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12395 • Publication Date (Web): 17 Oct 2017 Downloaded from http://pubs.acs.org on October 22, 2017

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ACS Applied Materials & Interfaces

Bio-inspired Antioxidant Defense System Constructed by Antioxidants-Eluting Electrospun F127-based Fibers Haozheng Wang,†,‡ Xiaodong Xu,§ Runhai Chen,† Jiruo Zhao,‡,* Lele Cui, § Guangkuo Sheng, § Qiang Shi, †,* Shing-Chung Wong, ┴ Jinghua Yin † †

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ‡

Shandong Provincial Key Laboratory of Olefin Catalysis and Polymerization/Key Laboratory of Rubber-Plastics (QUST), Ministry of Education/Shandong, Qingdao 266042, P. R. China

§

Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education, College of Materials Science and Chemical Engineering, Harbin Engineering University, Harbin 150001, China. ┴

Department of Mechanical Engineering, University of Akron, Akron, Ohio 44325-3903, USA Keywords: Antioxidant defense system, oxidative injure, Pluronic F127, red blood cells,

reactive electrospinning

ABSTRACT: Cells were continuously exposed to oxidative damage by overproduction of reactive oxygen species (ROS) when they contacted implanted biomaterials. The strategy to prevent cells from oxidative injures remains challenge. Inspired by the antioxidant defense system of cells, we constructed a biocompatible and ROS-responsive architecture on the substrate of styrene-b-(ethylene-co-butylene)-b-styrene elastomer (SEBS). The strategy was based on fabrication of architectures through reactive electrospinning of mixture including SEBS, acylated Pluronic F127, copolymer of poly(ethylene glycol) diacrylate and 1,2ethanedithiol (PEGDA-EDT) and antioxidants (AA-2G), and ROS-triggered release of AA2G from microfibers to detoxify the excess ROS. We demonstrated that the stable and hydrophilic architecture was constructed by phase separation of SEBS/F127 components and crosslinking between polymer chains during electrospinning; the ROS-responsive fibers controlled the release of AA-2G and the interaction of AA-2G with ROS reduced the 1 ACS Paragon Plus Environment


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oxidative damage to cells. The bio-inspired architecture not only reduced mechanical and oxidative damage to cells but maintained normal ROS level for physiological hemostasis. This work provides basic principles to design and develop anti-oxidative biomaterials for implantation in vivo.

1. INTRODUCTION The foreign body response to biomaterials is a cascade of events caused by implantation, followed by protein adsorption, blood cell adhesion and activation of immune cells, and ultimately recruitment of fibroblasts to form fibrous capsules.1-3 During this process, reactive oxygen species (ROS) are released by activated phagocytes including neutrophils and macrophages.4,

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ROS encompass a wide variety of diverse chemical species including

superoxide, hypochlorite, hydroxyl anions, and hydrogen peroxide.6-8 Chronically high levels of ROS induce damage to lipids, proteins, and DNA, and are involved in a number of pathological conditions including cancer, and neurodegenerative and cardiovascular disease. 911

Therefore, construction of biointerfaces on implant biomaterials to prevent the excess

production of ROS is highly desired. Current strategy to prevent ROS overproduction mainly depends on the introduction of anti-oxidant to the biological system with nanoparticles, microspheres, and polymer microcapsular.

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However, these methods do not work in constructing the anti-oxidative surface

on the implant biomaterials. On the one hand, the introduction of nanoparticles and microcapsular usually causes poor biocompatibility and the leakage of nanoparticles and microcapsular further leads to toxicity to cells.

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On the other hand, the anti-oxidants are not well

capsulated in the substrate, which renders the anti-oxidants sensitive to oxidation to lose longterm anti-oxidative capability.

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In addition, the release of anti-oxidative agents is not

responsive to ROS concentration, which breaks the ROS homeostasis to impair physiological 2 ACS Paragon Plus Environment

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function of body. It is well known that ROS within the certain concentration plays key roles in regulating cellular signaling pathways, combating pathogens and inducing oxidative degradation of biomaterials.

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Lowering ROS levels below the homeostatic set point

interrupts the physiological role of oxidants in cellular proliferation and host defense. 18 Up to now, the anti-oxidative surfaces on the implanted biomaterials are not constructed successfully. The organism has established the effective cellular strategies to detect and detoxify ROS. Through the signaling pathways including the extracellular signal-regulated kinase (ERK), cJun amino-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling cascades, the organism responds to ROS level sensitively and activates antioxidant defense system to resist the oxidative stress.

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A sophisticated enzymatic and non-

enzymatic antioxidant defense system includes catalase, superoxide dismutase, glutathione peroxidase, peroxiredoxins, ascorbate, pyruvate, flavonoids and glutathione, which counteracts and regulates overall ROS levels to maintain cellular physiological hemostasis. 21, 22

Inspired by the anti-oxidant defense system of cell/organism to ROS, we construct the

micro-fibrous architecture on SEBS substrate by encapsulation of antioxidant in ROSresponsive microfibers. The microfibers are prepared by one-step electrospinning of SEBS, acylated Pluronics 127 (A-F127), copolymer of poly(ethylene glycol) diacrylate (PEGDA) and 1, 2-ethanedithiol (EDT) (PEGDA-EDT) and antioxidant (AA-2G). The extremely large surface area and porosity of microfibers enhance the sensitivity of microfibers to excess ROS. The hydrophilicity and enhanced sensitivity of fibers enable precise manipulation of surface properties and create the opportunity to control anti-oxidant release. SEBS is selected as the substrate because it is biocompatible and used for implanted devices. 23,24 However, SEBSbased biomaterials sometimes induce oxidative damage to cells.16 Thus, developing the SEBS-based biomaterials with long-term anti-oxidative capability is a pressing task. SEBS is 3 ACS Paragon Plus Environment


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also selected as the electrospinning component to enhance the fiber stability on the SEBS substrate because of the same materials. Pluronics 127 are amphiphilic, triblock copolymers composed of poly(oxyethylene)-block-poly(oxypropylene)-block-poly-(oxyethylene), which has been approved by Food and Drug Administration (FDA) of USA for use as food additives and pharmaceutical ingredients.

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The presence of F127 renders the resulted fibers

hydrophilic and biocompatible due to the self-assembly of SEBS and F127 during electrospinning.

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PEGDA-EDT copolymer is the ROS-responsive polymer that is

sensitive to the excess ROS. The antioxidant, AA-2G is the derivative of L-ascorbic acid (LAA) but more stable than L-AA.

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However, AA-2G tends to be oxidized in the solution.

Therefore, encapsulation of AA-2G in the electrospun fibers is an effective method to prevent AA-2G from oxidation before its usage. Here, the bio-inspired anti-oxidant defense systems are fabricated on the SEBS substrate with ROS-responsive microfibers. The strategy is based on fabrication of stable SEBS/AF127/PEGDA-EDT/AA-2G fibers by reactive electrospinning, generation of hydrophilic and biocompatible surface through phase-separation of microfibers, and the release of AA-2G from the microfibers in response to the excess ROS. We demonstrate that the stable and biocompatible architecture is constructed by phase-separation of SEBS/A-F127 components and crosslinking between polymer chains during electrospinning; the electrospun fibers are responsive to the excess ROS and able to controll AA-2G release; the interaction of AA-2G with ROS reduces the oxidative damage to cells. Thus, the bio-inspired architecture reduces both mechanical and oxidative damage to cells. This work provides basic principles to design and develop anti-oxidative biomaterials for implantation in vivo.

2. EXPERIMENTAL SECTION 2.1. Materials 4 ACS Paragon Plus Environment

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SEBS copolymer with 29 wt% styrene (Kraton G 1652) was provided by Shell Chemicals. Block copolymer PEO-PPO-PEO (Pluronic F127) with an average molecular weight of 12600 g/mol and 73.2 wt% of ethylene glycol blocks was purchased from SigmaAldrich. Poly (ethylene glycol) diacrylate (PEGDA with average Mn =1K g/mol) was obtained from Sigma-Aldrich. 1, 8-Diazabicyclo [5.4.0] -7-undecene (DBU, 98%) was purchased from Adamas Reagent Ltd.

Benzophenone (BP) was provided by Peking Ruichen Chemical

(China). 2-O-α-D-Glucopyranosyl-L-ascorbic Acid (AA-2G, Mw = 338.27 g/mol) was supplied by Tokyo Chemical Industry (Japan). Chloroform, dimethylformamide (DMF), trimethylamine, acrylamide, methylene chloride, acetone and xylene were reagent grade products. Other reagents were AR grade and used without further purification. Phosphatebuffered saline (PBS 0.9% NaCl, 0.01M phosphate buffer, PH 7.4) was prepared freshly. 2.2 Synthesis of Acylated Pluronic F127 (A-F127) A-F127 was synthesized by conjugating acrylate onto the terminal hydroxyl groups of Pluronic F127. In brief, 2 g of F127 was dissolved in 50 mL of solvent of dichloromethane and toluene (2/1 wt%) with over-flowing of argon gas to displace oxygen. The solution was further degassed through three freeze-pump-thaw cycles, followed by addition of 0.3 g trimethylamine. The solution was added dropwise to 10 mL of methylene chloride containing acryloyl chloride (200 mg) for 30 min and reacted for 24 h in the dark. Finally, the acylated F127 was extracted against NaCl-saturated double distilled water, precipitated in ice-cold (~20 °C) diethyl ether, and dried under vacuum. The degree of acylation by comparing the relative peak area of a vinyl group (2H, CH2=, δ = 5.8~6.6, Chloroform-d) in acryloyl chloride and a methyne group (H, >CH-CH3, δ = 3.21, Chloroform-d) of mono propylene oxide in F127 of 1H-NMR. The degree of resulting acylation was 75~85%. The acylation of F127 did not affect its hydrophilicity.

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2.3 Synthesis of PEGDA-EDT and Oxidation of PEGDA-EDT The synthesis of PEGDA-EDT polymer was carried out by a thiol-ene reaction. In brief, EDT dithiol and PEGDA diacrylate were first dissolved in chloroform at an imbalance molar ratio of 120% under Ar gas for about 10 min and gentle stirring. Then the catalyzer DBU was added to promote the polymerization, which was conducted at room temperature for 15 h under Ar atmosphere. The synthesized polymer was precipitated by an excess amount of icecold ethyl ether three times and dried under vacuum for 48 h at room temperature. The monomers and synthesized polymer were characterized by 1H-NMR spectrometer (Bruker AV 400 MHz) in CDCl3. The oxidation of PEGDA-EDT polymer was studied by NMR spectrometer. Briefly, 600 µL of concentrated PEGDA-EDT solution (10.0 mg mL−1 in D2O) was first placed in the NMR tube and kept at 4 °C for 15 min, then 50 µL of H2O2 solution in D2O was added. Three concentration of H2O2 in the tube (H2O2/sulfur molar ratio of 3.0, 2.0 and 1.0, respectively) were studied. And the spectra of the mixture were then recorded at different time intervals. 2.4 Electrospinning of Fibers A mixture of chloroform and toluene

(80/20 wt %) was used as the solvent and

incorporated into a previously weighed dry mixture of SEBS/A-F127/ PEGDA-EDT/AA-2G at different ratios, with a constant total concentration polymer/solution of 17 wt %. Then, 2 mL ethanol solution containing 1.5 wt% BP and 1.2 wt% PEGDA was dropped in 4 mL mixed solution of chloroform and toluene. Finally, the mixed solutions were transferred to a syringe for electrospinning under UV irradiation. Four compositions were studied: SEBS, SEBS/AF127 (80/20 wt%), SEBS/A-F127/PEGDA-EDT (70/20/10 wt%) and SEBS/A-F127/PEGDAEDT/AA-2G (60/20/10/10 wt%). For comparison, the non-crosslinked fibers of SEBS/AF127/PEGDA-EDT/AA-2G (60/20/10/10 wt%) were prepared without UV irradiation. Considering induction with water renders the electrospun fibers hydrophilic,

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the

micro/nanofibers were electrospun at 50% relative humidity (RH). The micro/nanofibers were 6 ACS Paragon Plus Environment

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electrospun onto the surface SEBS at a room temperature, 50 % RH, solution feed rate 1.5~3 ml/h with applied voltage of 10-11 kV. The distance between needles and collector was ~15 cm. Some as-electrospun fibers were immerged in the distilled water for 3 to 24 h to confirm the reactions during electrospinning. For simplicity, the SEBS coated with electrospun fibers was referred as ‘electrospun SEBS’. The morphology of electrospun SEBS was characterized by a field-emission scanning electron microscopy (SEM, Sirion-100, FEI, USA) and a transmission electron microscopy (TEM JEM1011, Japan). During electrospinning, the TEM copper grids were used to collect electrospun fibers for 20 s to prepare samples for TEM measurement. These samples were dried at vacuum. Then, some samples were further treated for 5 min with water vapor from the hot distilled water (60 °C) and dried for TEM observation. Surface wettability of SEBS and electrospun SEBS (~300 µm thickness) was evaluated by the sessile drop method with a pure water droplet (ca. 3 µL) using a contact angle goniometer (DSA, KRUSS GMBH, Germany). The surface composition was determined via X-ray photoelectron spectroscopy (XPS) by using VG Scientific ESCA MK II Thermo Avantage V 3.20 analyzer with Al/K (hν =1486.6 eV) anode mono-X-ray source. 2.5 AA-2G release The electrospun meshes with size of 1 cm × 1 cm (100 mg) were incubated in PBS (11 mL) with the H2O2 in 0 mM, 5 mM and 10 mM, respectively. Then, at the desired time, 70 µL solution was collected and the amount of released AA-2G was measured using an instrument of TECAN (TECAN GENIOS, Austria) operating at 285 nm with a standard calibration curve. The release profile was normalized to the amount of AA-2G initially loaded in micro/nano fibers. 2.6 Determination of Hydrogen Peroxide Concentration of Model RBCs RBCs were used as model cells to evaluate the efficiency of bio-inspired anti-oxidant defense system. Fresh blood extracted from a healthy rabbit was immediately mixed with 3.8 7 ACS Paragon Plus Environment


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wt % sodium citrate solution at a dilution ratio of 9:1. (The experiments were carried out in accordance with the guidelines issued by the Ethical Committee of the Chinese Academy of Sciences. The Committee approved the experiments and informed consent was obtained for any experimentation with human subjects). Then the blood sample was centrifuged at 1000 rpm for 15 min to separate platelet rich plasma (PRP), white blood cells and red blood cells (RBCs). To mimic the long-term contact of blood with implanted devices, RBCs were stored in vitro in control SEBS bag and electrospun SEBS bags, whose surfaces were coated with different as-electrospun fibers. The determination of hydrogen peroxide concentration in the preserved RBCs was based on that hydroperoxides can form a steady orange peroxo-titanium complex with titanium (Ti4+) in alkaline solution and the complex can be dissolved in a sulphuric acid for further measurement by spectrophotometry. Peroxides were extracted by adding 50 µL preserved RBCs in 1 mL cold acetone and centrifuged (8000 rpm, 10 min, 4°C) to get 250 µL peroxide supernatant. Then 25 µL of a titanium reagent (50 mg/L titanic sulfate in concentrated HCl, w/v) were added，followed by the addition of 50 µL concentrated NH3•H2O to precipitate the peroxide-titanium complex. The precipitate washed repeatedly with acetone three times and solubilized in 250µL of 2M H2SO4, the absorbency of the obtained solutions was measured by TECAN absorbance reader (TECAN GENIOS, Austria) at 415 nm. A H2O2 standard curve was established between 0 and 10 mmol/L, following the same process used for sample determination. 2.7 Oxidative Damage of RBCs The oxidation damage of RBC was evaluated by the commercial FTIC Annexin V (A5) Apoptosis Detection Kit I. A5 was a 35-36 kDa Ca2+-dependent phospholipid-binding protein that had a high affinity for phosphatidylerine (PS), and bound to cells with exposed PS. Fluorescein isothiocyanate-labeled A5 (FITC-A5), retained its high affinity for PS and thus served as a sensitive probe for PS exposure and cell oxidation. The preserved RBCs were 8 ACS Paragon Plus Environment

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washed twice with cold PBS and then suspended in 1X Binding Buffer [0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2.] at a concentration of 1 × 106 cells/mL. Then, 100 µL of the solution (1 × 105 cells) was transfer to a 5 ml culture tube and 5 µL of FITC Annexin V was added to the tube, followed by gently vortex and incubation for 15 min at 25°C in the dark. Finally, the incubated cells were dropped onto poly-L-lysine-coated glass slides to adhesion for 30 min at 25°C in the dark, and visualized by CLSM at 488 nm within 1 h. The fluorescence, light field and merged images were obtained. 2.8 Hemolysis Assay and Mechanical Fracture of RBCs 0.1 mL preserved RBCs were collected in 2 mL PBS and centrifuged (3000 rpm, 3 min) to get the supernatant, and then it was transferred to 96-well plates. Positive and negative controls were produced by adding 0.1 mL of fresh RBCs to 2 mL of distilled water and normal saline, respectively. After incubation for 2 h, the RBCs were removed by centrifugation (3000 rpm, 3 min) and the supernatant was transferred to 96-well plates. Optical density (OD) of the supernatant was measured with TECAN absorbance reader (TECAN GENIOS, Austria) at 541 nm. The hemolysis ratio (HR) was calculated according to the following formula: HR(%) =

ODtest - ODneg OD pos - ODneg

×100 100 ×

(1)

where ODtest is test sample absorbance value, ODpos, and ODneg are the positive (water) and negative (saline) control, respectively. Results were given as the mean of triplicate experiments and standard deviation. The mechanical fragility (MF) test was performed according to the method developed by Raval et al. 30 Briefly, 300 µL preserved RBCs were diluted with 6 mL normal saline and then averagely transfer to six tubes (1.8 mL), three of which contained two ellipsoid magnetic stirrers (6 mm ×10 mm) and three of which did not. The tubes with stirrers were strongly shaken on a thermostatic oscillator for 2 h, while the remaining tubes without stirrers were not 9 ACS Paragon Plus Environment


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shaken and served as control to ascertain the initial concentration of free hemoglobin (Hb) in each aliquot. After shaking, all tubes were centrifuged twice. For comparison, the MF tests for fresh RBCs were performed under the same conditions. In addition, 50 µL fresh RBCs were diluted with 1 mL distilled water and incubated for 2 h. The free Hb concentrations in the supernatants were determined by TECAN absorbance reader (TECAN GENIOS, Austria) at 541 nm. The MF index (MFI) was then calculated according to the following formula:

(2) where Hbshaken is the mean free Hb concentration in the supernatants of the shaken specimens, Hbcontrol is the average free Hb concentration in the supernatants of the control samples, and Hbaliquot is the average Hb concentration in the supernatants of 50 µL fresh RBCs in 1 mL distilled water after 2 h treatments. 2.9 Statistical Analysis The hemolysis and oxidation degree are given as means ± SD for the indicated number of fresh and preserved RBCs. Statistical analysis was performed using Origin Software, with post hoc analysis by Bonferroni’s multiple comparison tests when appropriate. Differences were considered statistically significant at P ≤0.05.

3. RESULTS AND DISCUSSION 3.1. Bio-inspired Anti-oxidant Defense System Cell oxidation leads to cell age and death. 31 As the response, the cells have evolved the antioxidant defenses system to detoxify the excess ROS. A sophisticated enzymatic and nonenzymatic antioxidant defense system including catalase, superoxide dismutase and glutathione peroxidase counteracts and regulates overall ROS levels to maintain physiological hemostasis (Figure 1A). Superoxide dismutase accelerates the conversion of superoxide to 10 ACS Paragon Plus Environment

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hydrogen peroxide, whereas catalase and glutathione peroxidase convert hydrogen peroxide to water. In addition, a variety of other non-enzymatic, low molecular mass molecules are important in scavenging ROS.

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These molecules include pyruvate, ascorbate, flavonoids,

carotenoids and glutathione. The intricate antioxidant defense system counteracts on the burden of ROS production and helps the cell and organism adapt to ROS damage. Inspired by the antioxidant defense system, we design the architectures that can detect the excess ROS and release antioxidant triggered by ROS (Figure 1B). The released antioxidants counteract the excess ROS and reduce the extracellular ROS level to protect cells from oxidative damage.

Figure 1. 3 D Antioxidant defense architectures composed of electrospun ROS responsive polymer (B) by the mimicry of antioxidant cellular defense system (A). Bio-inspired antioxidant defense system (ADS) can detoxify excess reactive oxygen species (ROS) and maintain ROS hemostasis.



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Figure 2. Schematic of constructing an antioxidative defense system on SEBS substrate by combined functional polymer synthesis and reactive electrospinning. The ROS responsive polymer (PEGDA-EDT) and acylated F127 (A-F127) are synthesized and mixed with SEBS and AA-2G in the mixture of chloroform and toluene for reactive electrospinning. The surface of resulting microfiber is hydrophilic and AA-2G can be released from the microfibers triggered by overproduction of ROS to pretect cells from oxidative damage.

The strategy takes advantages of fabricating stable SEBS/A-F127/PEGDA-EDT/AA-2G fibers by one-step reactive electrospinning, generation of hydrophilic and biocompatible surface through phase-separation of microfibers, and the release of AA-2G from the microfibers in response to excess ROS (Figure 2). Firstly, the ROS-responsive polymer (PEGDA-EDT) is synthesized by thiol-ene polymerization of PEGDA and EDT monomers. F127 acylation is performed by conjugating acrylate onto the terminal hydroxyl groups of Pluronic F127. Then, PEGDA-EDT and acylated F127 (A-F127) are mixed with SEBS and 12 ACS Paragon Plus Environment

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AA-2G for one-step electrospinning. Finally, the stable and biocompatible architectures are constructed on SEBS substrates. During electrospinning, phase separation of A-F127 and SEBS enables the fiber surface hydrophilic and subsequent reactions among A-F127, PEGDA-EDT and SEBS renders the as-electrospun fibers stable. The hydrophilic surface of A-F127 can form hydration layer with water to reduce the impact on cells and prevent cell adhesion on the fiber surface when the cells colloid with fibers. Furthermore, the switch from hydrophobicity to hydrophilicity of PEGDA-EDT in response to the excess ROS facilitates the release of AA-2G. As a result, the released AA-2G interacts with ROS to reduce the excess ROS, protecting cells from oxidative damage and maintaining physiological hemostasis.

3.2. Synthesis of A-F127, PEGDA-EDT and Oxidation of PEGDA-EDT F127 acylation is performed by conjugating acrylate onto the terminal hydroxyl groups of Pluronic F127 (Supporting Information, Figure S1). The successful acylation is confirmed by 1H-NMR spectra (Figure 3A). The degree of acylation is determined by comparing the relative peak area of the acryl protons of A-F127 (5.8~6.6 ppm) with that of three protons of the methyl group in the propylene oxide unit (1.0 ppm) in 1H-NMR spectra. 32 The degree of the resulting acylation is 75% ~ 85%. The unsaturated groups on the terminal of A-F127 provide the active sites for reactive electrospinning. The synthesis of PEGDA-EDT polymer is carried out by a click thiol-ene reaction. The monomers and synthesized polymer are characterized by 1H-NMR in CDCl3 (Figure 3B). The peaks at δ = 2.88-2.78 ppm and 2.702.60 ppm corresponding to the ester groups in the linkage of hydrophilic and hydrophobic segments are observed in the spectrum of synthesized polymer, demonstrating the successful thiol-ene polymerization and formation of periodical β-thiopropionate group in backbone.

33

The resulting PEGDA-EDT copolymers exhibit oxidation sensitive due to the presence of oxidizable thioether groups in the backbone.



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Figure 3. 1H-NMR spectra of acylated F127 (A), PEGDA-EDT copolymer (B) and oxidation of PEGDA-EDT copolymer with H2O2 (C), and the normalized extents of oxidation as functions of time at different H2O2/sulfur molar ratios (D).

The oxidation behavior of PEGDA-EDT copolymers is detected in situ by 1H-NMR measurements. Figure 3C shows the time dependent 1H-NMR spectra of PEGDA-EDT in the presence of four equivalents of H2O2 per sulfur atom in D2O. Two multiple peaks at 2.95-3.15 and 3.15-3.35 ppm, presumably ascribed to methylene groups in the β position of sulfoxides (e′+f′) and sulfones (e′′ +f′′), respectively, rise gradually upon addition of H2O2. Meanwhile, the gradual disappearance of methylene e and f at 2.68-2.80 ppm and the sift of methylene d (14 ACS Paragon Plus Environment

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OC(O)CH2-) from 2.58-2.68 to 2.78-2.89 ppm further verifies the oxidation of sulfur atoms by H2O2.34 The collapsed polymers convert water-soluble polymers by oxidative conversion of hydrophobic thioether groups into hydrophilic sulfoxide and sulfone groups.35 The extents of oxidation are quantitatively analyzed by plotting the integrals of methylene e′+f′+ e′′ +f′′ against methylene c (Figure 3D). The extent of oxidation is calculated to be 95, 83, and 45% for PEGDA-EDT after 120 h incubation with H2O2 at H2O2/sulfur molar ratio of 3.0, 2.0 and 1.0, respectively. The oxidation-switchable water solubility is used to design oxidationtriggered antioxidant delivery to maintain the ROS balance in the system.

3.3. Architecture Construction and Surface Properties SEBS, A-F127, PEGDA-EDT and AA-2G are mixed in the solvent of chloroform and toluene. Then, ethanol solution containing 1.5 wt% BP and 1.5 wt% PEGDA is added to the mixture for electrospinning at 50% relative humidity (RH) and with the UV irradiation. The phase separation and subsequent UV-induced reactions occur during the electrospinning process.36,

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To check the phase separation, SEBS and SEBS/A-F127 (80/20 wt) are

electrospun under the same conditions, respectively. The electrospun SEBS microfibers exhibit varied diameter and hydrophobicity with the water contact angle (WCA) of 112° (Figure 4a). In the presence of A-F127, the microfibers become uniform and no beads are observed (Figure 4b). The surface is hydrophilic with the WCA of 18°. The phase separation between SEBS and A-F127 is the main reason for the hydrophilicity change. 27, 28, 38 The XPS analysis verifies the phase separation (Figure 4d). Compared with the C 1s spectrum (a single peak at 284.5 eV) of the control SEBS fibers (inset of Figure 4d), the C 1s spectrum of SEBS/A-F127 electrospun fibers shows three peaks at 284.5, 286.2 and 288.7 eV, ascribed to the C-H, C-O- and O=C-O species of A-F127, respectively.

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Based on the ratio of oxygen

content to carbon and oxygen content {[O]/([C]+[O])} in the electrospun fibers, 39 the content 15 ACS Paragon Plus Environment


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of A-F127 on the fiber sheath is calculated to be 75%. The phase separation occurs because of immiscible blends of SEBS and A-F127 and different flow viscosity of each component during electrospinning.28, 38

Figure 4. Images and surface properties of electrospun fibers. SEM images of the electrospun fibers of (a) SEBS, (b) SEBS/A-F127, (c) SEBS/A-F127/PEGDA-EDT/AA-2G. (d) C1s corelevel spectrum of SEBS/A-F127 and (e) TEM image of electrospun fiber after induction with water vapor for 5 min, (f) SEBS/A-F127/PEGDA-EDT/AA-2G after immersion in distilled water for 3 h. The inset in Figure 4e is TEM image of electrospun SEBS/A-F127 fibers. The arrows in Figure 4c indicate the bead formation on the fibers.

The presence of PEGDA-EDT has slight effect on fiber morphology and surface hydrophilicity. Encapsulation of AA-2G in the fiber results in two types of fibers: thicker fibers without beads and thinner fibers with beads (Figure 4c). The fiber morphology is related to the non-homogenous distribution of AA-2G in the solution and jet instability of polymer solution. 40 The fiber surface remains hydrophilic with WCA of 8°. The morphology 16 ACS Paragon Plus Environment

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of electrospun fibers is further observed with TEM (Figure 4e). SEBS/A-F127 fiber (inset of Figure 4e) exhibits obvious core-shell structure, providing the direct evidence for the phase separation. Based on the data of WCA and XPS analysis (Figure 4a-d), A-F127 is determined to cover the fiber surface. To observe all phases of electrospun fibers, SEBS/A-F127/PEGDAEDT/AA-2G fibers are electrospun without UV irradiation and these non-crosslinked fibers are treated with water vapor before TEM measurement. The scatter distribution of AA-2G on the fiber surface and the vague border along the fiber are observed. The vague border is mainly caused by the leakage of A-F127 from the fiber after induction with water vapor. As PEGDA-EDT has the similar unit structure (-CH2CH2O-) as that of A-F127, it tended to localize in the A-F127 phase. The existence and distribution of each component in the electrospun fibers is further supported by the XPS analysis on the samples of SEBS/A-F127 and SEBS/A-F127/PEGDA-EDT/AA-2G, respectively (Supporting Information, Figure S2). The setup for electrospinning is designed to induce phase separation of SEBS and A-F127 firstly, followed by the UV-induced reactions (Supporting Information, Figure S3). The UVinduced reactions include PEGDA-mediated binding between A-F127 and SEBS and coupling reactions between PEGDA-EDT and A-F127 through thiol-ene reactions (Supporting Information, Figure S4). In contrast to crosslinking after electrospinning, reactive electrospinning does not induce cytotoxicity or need additional processing step.

36, 37

The

produced microfibers are stable under physiological conditions. To check the stability of the crosslinked fibers of SEBS/A-F127/PEGDA-EDT/AA-2G, the electrospun fibers are immersed in distilled water for 3 h. As shown in Figure 4f, the surface of fibers becomes rough because of the presence of porous structure, but the architecture on SEBS does not collapse, indicating most A-F127 maintains in the electrospun fibers. The stability of electrospun fibers is further supported by the slight change of oxygen content after immersion of fibers in distilled water for 24 h (Supporting Information, Figure S5). The stable and hydrophilic surface renders the fiber biocompatible and reduces the mechanical damage to the 17 ACS Paragon Plus Environment


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cells. 16 The protein adsorption and platelet adhesion are performed on the electrospun fibers. Compared with electrospun SEBS fibers, the platelet adhesion and protein adsorption are reduced substantially on the electrospun SEBS/A-F127/PEGDA-EDT/AA-2G fibers (Supporting Information, Figure S6). The cytotoxicity of electrospun fibers to L929 murine fibroblast cells is tested. The cell viability after treatments with electrospun fibers is higher than 80% (Supporting Information, Figure S7), indicating high biocompatibility of the electrospun fibers.

3.4. Anti-oxidation of Electrospun Meshes ROS-triggered release of anti-oxidant and its reaction with ROS are essential for successful anti-oxidant defense system. AA-2G is the derivative of L-ascorbic acid (L-AA), which has drawn great interest for applications in the cosmetic, food, and healthcare areas. 29 It exhibits highly reactive to ROS to reduce the oxidative damage of cells. The release of AA-2G in PBS buffer is shown in Figure 5A. Without H2O2, the relase of AA-2G is slow and only about 30% loaded AA-2G is released after 5 days (Figure 5A-a). The AA-2G release becomes fast in the presence of H2O2 and nearly 60% and 70% loaded AA-2G are released with 5 mmol/L and 10 mmol/L of H2O2, respectively (Figure 5A-b,c). PEGDAEDT copolymers are easily converted into completely water-soluble polymers by oxidative conversion of hydrophobic thioether groups into hydrophilic sulfoxide and sulfone groups. The hydrophilic groups of electrospun fibers tend to adsorb water molecules to behavior like hydrogels because of crosslinked structure in fibers. As the result, the swelled hydrogels facilitate fast and high-content release of AA-2G. 33, 41


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Figure 5. Anti-oxidant defense of bio-inspired system (A) H2O2 induced AA-2G release. a) without H2O2, b) with 5 mmol/L, c) with 10 mmol/L; (B) H2O2 concentration of storaged RBCs. a) SEBS bags, b) SEBS/A-F127 bag, c) SEBS/AF127/PEGDA-EDT bags, d) SEBS/A-F127/PEGDA-EDT/AA-2G bags. (C) Fluroscence and merged images of RBCs. a), b) fluroscence and merged images of RBCs in SEBS bag; c), d)

fluroscence and merged images of RBCs in SEBS/A-

F127/PEGDA-EDT/AA-2G bag; (D) Hemolysis and mechanical fragility index of RBCs.

To check the efficiency of anti-oxidant defense system, red blood cells (RBCs) are selected as model cells for investigation. RBCs play key roles in transportation of oxygen/nutrients, blood-based detection and immunoprotection.

16

To mimic the long-term

contact of implanted device with blood in vitro, RBCs are packaged in the bags made of 19 ACS Paragon Plus Environment


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SEBS film and electrospun SEBS and preserved at 4 °C for 5 days (Supporting Information, Figure S8). The accumulation of hydrogen peroxide under different conditions is determined by spectrophotometry (Supporting Information, Figure S9).

42

Due to metabolism and

oxidation stress from control SEBS bags, the H2O2 concentation increases fastly and reaches about 1.0 mmol/L and 2.0 mmol/L at the first day and 5th day, respectivley (Figure 5B-a). The similar tendency has been observed by D’Alessandro et al., who reported that high amount of H2O2 accumulation in the preserved RBCs. 43 In most cases, biologically relevant equilibrium H2O2 levels are in the range from 5 µmol/L to 200 µmol/L, 44,45 the high H2O2 concentration indicates serious oxidation of RBCs. Reduced H2O2 concentration is observed in SEBS/A-F127 bag, but the H2O2 concentration is nearly 1.0 mmol/L after 5-day stroage (Figure 5B-b), confirming surface modification with hydrophilic layer has slight effect on cell oxidation.

16

H2O2 concentration decreases further in SEBS/A-F127/PEGDA-EDT bag due to

the H2O2 consumption by the reaction between H2O2 and PEGDA-EDT (Figure 5B-c). H2O2 conentration remains biologically relevant level (~200 µmol/L) in the bag of SEBS/AF127/PEGDA-EDT/AA-2G even after 5-day storage (Figure 5B-d), which confirms that released AA-2G interacts with ROS and decreases the H2O2 conentration substantially. In addition, regualtion of H2O2 conentration in biological level helps the cells for normal function. The oxidation damage to RBCs is evaluated by phosphatidylerine (PS) concentration on the cell surface with fluorescein isothiocyanate (FITC)-labeled Annexin V (FITC-A5). In oxidative and apoptotic cells, the membrane phospholipid PS translocates from the inner to the outer leaflet of the plasma membrane.

31

Because Annexin V (A5) has a high affinity for

PS, FITC-A5 serves as a sensitive probe for PS exposure. Fluorescent and merged images of preserved RBCs after PS-A5 binding are shown in Figure 5C. Bright fluorescence on the cell surface is oberved for RBCs in the SEBS bags (Figure 5C-a,b), indicating the serious cell 20 ACS Paragon Plus Environment

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oxidation. In contrast, the fluorescence can not be detected for the cells in the SEBS/AF127/PEGDA-EDT/AA-2G bags (Figure 5C-c,d), illustrating the SEBS/A-F127/PEGDAEDT/AA-2G bags has the extra anti-oxidative ability. The hemolysis and mechanical fragility index (MFI) of RBCs in different bags are shown in Figure 5D. After 5-day storage, the hemolysis ratio and MFI of preserved RBCs decreases in the order of bag of SEBS, SEBS/A-F127, SEBS/A-F127/PEGDA-EDT and SEBS/A-F127/PEGDA-EDT/AA-2G. The above results demonstrate PEGDA-EDT and released AA-2G reduce the oxidative injure to the RBCs. The advantages of anti-oxidant defense system are further demonstrated by the morphology observation of RBCs (Supporting Information, Figure S10) and data analysis of oxidation degree and normal shape ratio (Supporting Information, Figure S11). The method is facile and versatile, which paves new way to prepare the anti-oxidative implant biomaterials in vivo.

4. CONCLUSION In summary, inspired by the antioxidant defense system, we constructed the hydrophilic architecture on the SEBS substrate with ROS responsive microfibers. The strategy was based on fabrication of stable SEBS/A-F127/PEGDA-EDT/AA-2G fibers through reactive electrospinning, generation of hydrophilic surface through phase separation of microfibers, and the release of AA-2G controlled by ROS-responsiveness of microfibers. We demonstrated that the stable and hydrophilic architecture was constructed by phase separation of SEBS/F127 components and interactions between polymer chains during electrospinning; the controlled release of AA-2G was achieved by ROS-responsiveness of fibers and the interaction of AA-2G with ROS reduced the oxidative damage to cells. Thus, the bio-inspired architecture not only reduced mechanical and oxidative damage to cells but regulated the H2O2 concentration in biological level, which was essential to maintain physiological



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hemostasis for cells. This work provided basic principles to design and develop anti-oxidative biomaterials for future implants in vivo.

ASSOCIATED CONTENT Supporting Information Reaction pathway for F127 acylation; Reaction pathway for F127 acylation; XPS analysis the component in the electrospun fibers; Main reactions during electrospinning; Oxygen content in electrospun fibers; Protein adsorption and platelet adhesion; Cytotoxicity of electrospun fibers; RBCs preservation; Standard curve for H2O2 determination; Morphology of preserved RBCs; Oxidation degree and normal morphology. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *Tel: +86 431 85262109. Fax: +86 431 85262109. *E-mail: [email protected] ; [email protected]

Notes The authors declare no competing ﬁnancial interest.

Acknowledgements This work was supported by the financial support of the National Key Research and Development Program of China (2016YFC1100402), the National Natural Science Foundation of China (51573186), and Open Research Fund of State Key Laboratory of Polymer Physics and Chemistry, CIAC, CAS (201620 and 201628).


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1 Bio-inspired Antioxidant Defense System Constructed by

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